Automated Pipetting Apparatus Having a Combined Liquid Pump and Pipette Head System

ABSTRACT

The technology described herein generally relates to systems for extracting polynucleotides from multiple samples, particularly from biological samples, and additionally to systems that subsequently amplify and detect the extracted polynucleotides. The technology more particularly relates to microfluidic systems that carry out PCR on multiple samples of nucleotides of interest within microfluidic channels, and detect those nucleotides. The technology still more particularly relates to automated devices for carrying out pipetting operations, particularly on samples in parallel, consistent with sample preparation and delivery of PCR-ready nucleotide extracts to a cartridge wherein PCR is run.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/173,023, filed by ExpressMail on Jul. 14, 2008 (and entitled“Integrated Apparatus for Performing Nucleic Acid Extraction andDiagnostic Testing on Multiple Biological Samples”, in the name ofWilliams, et al.), which claims benefit of priority to U.S. provisionalpatent application Ser. No. 60/959,437, filed Jul. 13, 2007, both ofwhich are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The technology described herein generally relates to systems and methodsfor controlling fluid processing operations associated with extractingpolynucleotides from samples, particularly multiple biological samplesin parallel. The technology more particularly relates to automatedpipetting systems that operate in conjunction with reagent containersand carry out various suck and dispense operations on various reagentsin the containers, thereby bringing about mixing or disposal of thosereagents.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss or mishandling. Second, the equipment inquestion is typically not available ‘on-demand’ but instead runs inbatches, thereby delaying the processing time for many samples becausethey must wait for a machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector of interest. Onceamplified, the presence of a nucleotide of interest from the sampleneeds to be determined unambiguously. Preparing samples for PCR iscurrently a time-consuming and labor intensive step, though not onerequiring specialist skills, and could usefully be automated. Bycontrast, steps such as PCR and nucleotide detection have customarilyonly been within the compass of specially trained individuals havingaccess to specialist equipment.

Sample preparation is labor intensive in part because of the number ofreagents required, and the need for multiple liquid transfer (e.g.,pipetting) operations. Thus, there is a need for an automated pipettingapparatus, particularly one that can operate on multiple samples inparallel.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as at the priority date of anyof the claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The technology herein includes a liquid dispenser, comprising: one ormore sensors; a manifold; one or more pumps in fluid communication withthe manifold; one or more dispense heads in fluid communication with themanifold; and electrical connections that accept electrical signals froman external controller, wherein the liquid dispenser has no inlet oroutlet for fluids, other than through the one or more pumps. The liquiddispenser further has a number of dispense heads, wherein each head isconfigured to accept a pipette tip.

The technology herein further includes an automated pipetting systemthat includes a liquid dispenser, the dispenser comprising: one or moresensors; a manifold; one or more pumps in fluid communication with themanifold; one or more dispense heads in fluid communication with themanifold; and electrical connections that accept electrical signals froman external controller, wherein the liquid dispenser has no inlet oroutlet for fluids, other than through the one or more pumps.

The technology herein further includes an apparatus for carrying outsample preparation on multiple samples in parallel, the apparatusincluding an automated pipetting system configured to carry out liquidhandling steps associated with sample preparation. The pipetting systemincludes a liquid dispenser, the dispenser comprising: one or moresensors; a manifold; one or more pumps in fluid communication with themanifold; one or more dispense heads in fluid communication with themanifold; and electrical connections that accept electrical signals froman external controller, wherein the liquid dispenser has no inlet oroutlet for fluids, other than through the one or more pumps. Theapparatus may further carry out diagnostic analysis on nucleotides putinto form ready for amplification after sample preparation, where theautomated pipetting system is configured to transfer those samples to adevice that can amplify those samples and provide detectable quantitiesof amplified samples.

The technology herein further includes methods of sample preparation,comprising liquid handling steps that are performed on multiple samplesin parallel by an automated pipetting system that includes a liquiddispenser as further described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an automated apparatus configured to carryout sample preparation using a liquid dispenser as described herein.

FIGS. 2A and 2B show views of the exterior and interior of an exemplarydiagnostic apparatus.

FIGS. 3A and 3B show an exemplary embodiment of a reagent holder, inside plan, and perspective, views.

FIG. 4 shows a perspective view of a second exemplary embodiment of areagent holder, in perspective view.

FIG. 5 shows embodiments of a laminated structures used to seal liquidcontaining tubes.

FIG. 6 shows a sequence of pipetting operations in conjunction with alaminated layer, as in FIG. 5.

FIG. 7 shows perspective views of an exemplary rack for samples andreagent holders.

FIG. 8 shows perspective views of the rack of reagent holders and sampletubes of FIG. 7, in conjunction with a heater unit.

FIG. 9 shows a sequence of pipetting operations in conjunction with areagent tube.

FIG. 10 shows a side schematic view of a pipette head in position todispense liquid into a microfluidic cartridge.

FIG. 11 shows a block diagram of a liquid dispenser, showingcommunication between various components thereof.

FIG. 12 shows a liquid dispense head.

FIGS. 13A and 13B show views of a liquid dispenser.

FIGS. 14A-14C show views of a liquid dispense head.

FIG. 15 shows an exemplary distribution manifold.

FIGS. 16A and 16B show an exemplary device for stripping pipette tips.

FIGS. 17A-17C show three positions of a stripper/alignment plate duringoperation of a pipette tip stripper.

FIG. 18 shows a pipette tip stripper, and pipette tip sensors.

FIG. 19 shows a scanning read-head attached to a liquid dispense head,positioned over a number of reagent holders.

FIG. 20 shows a scanner in side view, positioned to read identifiers onone or more sample tubes.

FIG. 21 shows a scanner positioned above a microfluidic cartridge.

FIGS. 22A-22C show, schematically, pipette head usage during variouspreparatory processes.

Like reference numerals in the various drawings indicate like elements.

DETAILED DESCRIPTION

The automated pipetting apparatus described herein is typicallyconfigured for use in a method and apparatus for carrying out samplepreparation on biological samples in parallel, with or without PCR anddetection on the prepared samples, and preferably with high throughput.

Overview of a Preparatory or Diagnostic Apparatus that Incorporates aLiquid Dispenser

A schematic overview of an apparatus 981 for carrying out automatedsample preparation on multiple samples in parallel, according to stepsexemplified elsewhere herein, is shown in FIG. 1. The geometricarrangement of the components of system 981 is exemplary and notintended to be limiting.

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component requiring control, for examplevia a bus. It is to be understood that many such control functions canoptionally be carried out manually, and not under control of theprocessor. Furthermore, the order in which the various functions aredescribed, in the following, is not limiting upon the order in which theprocessor executes instructions when the apparatus is operating. Asuitable processor 980 can be designed and manufactured according to,respectively, design principles and semiconductor processing methodsknown in the art.

Processor 980 can be configured to accept user instructions from aninput device 984, where such instructions may include instructions tostart analyzing the sample, and choices of operating conditions.Processor 980 can be also configured to communicate with a display 982,so that, for example, information about an analysis is transmitted tothe display and thereby communicated to a user of the system. Suchinformation includes but is not limited to one or more of: the currentstatus of the apparatus; progress of PCR thermocycling; and a warningmessage in case of malfunction of either system or cartridge.Additionally, processor 980 may transmit one or more questions to bedisplayed on display 982 that prompt a user to provide input in responsethereto. Thus, in certain embodiments, input 984 and display 982 areintegrated with one another.

Processor 980 can be optionally further configured to transmit resultsof an analysis to an output device 986 such as a printer, a visualdisplay such as display 982 or a second display, a display that utilizesa holographic projection, or a speaker, or a combination thereof.Processor 980 can be still further optionally connected via acommunication interface such as a network interface to a computernetwork 988.

Processor 980 can be further configured to control various aspects ofsample preparation and diagnosis, as follows in overview. In FIG. 1, theapparatus 981 is configured to operate in conjunction with acomplementary rack 970. Apparatus 981 may be capable of receivingmultiple racks, such as 1, 2, 3, 4, or 6 racks.

Embodiments of rack 970 are further described in U.S. patent applicationSer. No. 12/173,023, filed by ExpressMail on Jul. 14, 2008 (and entitled“Integrated Apparatus for Performing Nucleic Acid Extraction andDiagnostic Testing on Multiple Biological Samples”, in the name ofWilliams, et al.), and Ser. No. 12/178,584, filed on Jul. 23, 2008, andentitled “Rack For Sample Tubes And Reagent Holders”, in the name ofDuffy, et al., both of which are incorporated herein by reference intheir entireties. A rack 970 is itself configured to receive a number ofbiological samples 996, such as nucleic-acid containing samples, in aform suitable for work-up and subsequent diagnostic analysis, and anumber of holders 972—as further described herein, such as in connectionwith FIG. 2—that are equipped with various reagents, pipette tips andreceptacles. The rack is configured so that, during sample work-up,samples are processed in the respective holders, the processingincluding being subjected, individually, to heating and cooling viaheater assembly 977.

The heating functions of the heater assembly 977 can be controlled bythe processor 980. Heater assembly 977 operates in conjunction with aseparator 978, such as a magnetic separator, that also can be controlledby processor 980 to move into and out of close proximity to one or moreprocessing chambers associated with the holders 972, wherein particlessuch as magnetic particles are present. Assembly 977 and separator 978are further described in U.S. patent application Ser. No. 12/178,586,filed on Jul. 23, 2008, and entitled “Integrated Heater and MagneticSeparator”, in the name of Handique, which is incorporated herein byreference in its entirety.

Processor 980 can be configured to receive data about a sample to beanalyzed, e.g., from a sample reader 990, which may be a barcode reader,an optical character reader, or an RFID scanner (radio frequency tagreader). Thus, sample reader 990 is configured to transmit identifyingindicia about the sample, and in some instances the holder, to processor980. In some embodiments, the sample reader is movable from one sampleposition to another. In some embodiments a sample reader is attached tothe liquid dispenser 976 and can thereby read indicia about a sampleabove which the liquid dispenser is situated. In other embodiments thesample reader is not attached to the liquid dispenser and isindependently movable, under control of the processor.

Liquid dispenser 976, which similarly can be controlled by processor 980and is further described herein, is configured to automatically carryout various pipetting (e.g., suck and dispense) operations on respectivesamples in rack 970, and fluids and reagents in the holders 972, toachieve extraction of nucleic acid from the samples. Liquid dispenser976 can carry out such operations on multiple holders simultaneously,and is further described herein.

Liquid dispenser 976 is also configured to take aliquots of fluidcontaining nucleic acid extracted from one or more samples and directthem to a storage area (not shown in FIG. 1), which may comprise acooler or coolers. Such a storage area may contain, for example, a PCRtube corresponding to each sample and which can contain solutions ofextracted nucleic acids dispensed by the liquid dispenser.

In the embodiment of a diagnostic apparatus shown in FIG. 1, a cartridge994 is received in bay 992. The receiving bay is in communication with aheater 998 that itself can be controlled by processor 980 in such a waythat specific regions of the cartridge 994 are heated at specific timesduring analysis. Liquid dispenser 976 is thus configured to takealiquots of fluid containing nucleic acid extracted from one or moresamples and direct them to one or more respective inlets in cartridge994. Cartridge 994 is configured to amplify, such as by providingchambers for carrying out PCR on, the respective nucleic acids.Exemplary cartridges are found described in U.S. patent application Ser.No. 12/173,023, filed Jul. 14, 2008, and incorporated herein byreference. The processor is also configured to control and receive datafrom a detector 999 that receives an indication of a diagnosis from thecartridge 994. The diagnosis can be transmitted to the output device 986and/or the display 982, as described hereinabove.

Embodiments of the apparatus shown in outline in FIG. 1, as with otherexemplary embodiments described herein, are advantageous because they donot require locations within the apparatus suitably configured forstorage of reagents. Therefore, the apparatus in FIG. 1 isself-contained and operates in conjunction with holders 972, wherein theholders are pre-packaged with reagents, such as in locations within itdedicated to reagent storage.

The apparatus of FIG. 1 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat they can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. Theapparatus is typically provided with a power-cord so that it can acceptAC power from a mains supply or generator. The apparatus may also beconfigured to operate by using one or more batteries and therefore isalso typically equipped with a battery recharging system, and variouswarning devices that alert a user if battery power is becoming too lowto reliably initiate or complete a diagnostic analysis.

The apparatus of FIG. 1 may further be configured, in other embodiments,for multiplexed sample analysis and/or analysis of multiple batches ofsamples, where, e.g., a single rack holds a single batch of samples.Each component shown in FIG. 1 may therefore be independently present asmany times as there are batches of samples (or some fraction thereof),though the various components may be configured in a common housing.

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis using the apparatus as further described herein caninclude one or more of the following steps: contacting a neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides (in some embodiments, the PCRreagent mixture can further include a positive control plasmid and afluorogenic hybridization probe selective for at least a portion of theplasmid); in some embodiments, the PCR reagent mixture can be in theform of one or more lyophilized pellets, as stored in a receptacle on aholder, and the method can further include reconstituting the PCR pelletwith liquid to create a PCR reagent mixture solution.

The apparatuses as described herein find application to analyzing anynucleic acid containing sample for any purpose, including but notlimited to genetic testing, and clinical testing for various infectiousdiseases in humans.

The apparatus herein can be configured to run on a laboratory benchtop,or similar environment, and can test approximately 45 samples per hourwhen run continuously throughout a normal working day. Results fromindividual raw samples are typically available in less than 1 hour.

FIGS. 2A and 2B show views of an exemplary diagnostic apparatus 3000incorporating various elements of FIG. 1. Shown in FIG. 2A, a front planview of apparatus 3000 has a hinged cover 3010, shown in a closedposition, bearing an optional clear window 3012 (that provides a userwith an at-a-glance indication of the operational state of theapparatus), and a handle 3014 that facilitates opening and closing ofthe cover.

Shown in FIG. 2B is a front plan view of apparatus 3000 with cover 3010moved to an open position revealing certain elements of the interior3020 of the apparatus. Aspects of the interior of the apparatus that arevisible in the view of FIG. 2B include: two removable racks 970, eachbearing 12 holders 972, and a liquid dispenser 976, mounted on a gantrythat can move along horizontal sliding rails 2102, as further describedherein.

Reagent Holders

The automated diagnostic apparatus described herein is configured tocarry out sample preparation on multiple samples by accessing more thanone sample tube, and more than one reagent holder, simultaneously. Thus,the liquid dispense head, further described herein, is configured toextract and dispense volumes of liquid from various positions in one ormore reagent holders, the holders being disposed in a suitablyconfigured rack, as also described elsewhere herein.

Described herein are reagent holders for holding and transportingreagents for various purposes, in particular sample preparation in aclinical context, and configured to be received by a rack as describedelsewhere herein. The reagent holders also typically provide acontainer, such as a process tube, in which various reagents can bemixed one with another and/or with a sample, and subjected to heating.

Exemplary reagent holders are further described in copending applicationSer. No. 12/218,416, filed by ExpressMail on Jul. 14, 2008 (and entitled“Reagent Tube, Reagent Holder, and Kits Containing Same”, in the name ofWilson, et al.) and incorporated herein by reference.

FIG. 3A shows a side plan view, and FIG. 3B shows a perspective view, ofan exemplary holder 804-1 as further described herein. This exemplaryholder, as well as others consistent with the written description hereinthough not shown as specific embodiments, are now described. FIG. 4shows a second embodiment of a reagent holder 804-2, in perspectiveview, the holder having a different configuration of containers fromthat in FIGS. 3A and 3B. Like reference numerals in FIGS. 3A, 3B, and 4refer to like elements in those respective figures. Holder embodiments804-1 and 804-2 may be referred to collectively, herein as holder 804.

The exemplary holders of FIGS. 3A, 3B, and 4 comprise a connectingmember 510 having one or more characteristics as follows. Connectingmember 510 serves to connect various components of the holder together.Connecting member 510 has an upper side 512 and, opposed to the upperside, an underside 514.

The reagent holder of FIGS. 3A, 3B, and 4 are configured to comprise: aprocess tube 520 affixed to the connecting member and having an aperture522 located in the connecting member; at least one socket 530, locatedin the connecting member, the socket configured to accept a disposablepipette tip 580; an optional pipette sheath 570 as further describedherein; two or more reagent tubes 540 disposed on the underside of theconnecting member, each of the reagent tubes having an inlet aperture542 located in the connecting member; and one or more receptacles 550,located in the connecting member, wherein the one or more receptaclesare each configured to receive a complementary container such as areagent tube (not shown in FIG. 3B) inserted from the upper side 512 ofthe connecting member. Each of the apertures, and the correspondingopenings of various complementary containers, is configured to accept apipette tip, such as a standard laboratory pipette tip, during variouspipetting operations such as dispensing fluid into, or sucking fluid outof, the one or more containers.

The one or more receptacles 550 are configured to accept container 554that contain, respectively, sufficient quantities of one or morereagents typically in solid form, such as in lyophilized form, forcarrying out extraction of nucleic acids from a sample that isassociated with the holder. The receptacles can be all of the same sizeand shape, or may be of different sizes and shapes from one another.Preferably the receptacles 550 are configured to accept commonly usedcontainers in the field of laboratory analysis, or containers suitablyconfigured for use with the holder herein. The containers may be snap-inreagent tubes that maintain a steady position in the holder duringpipetting operations thereon.

The containers that contain solid reagents such as lyophilized reagents,can be sealed across their tops by a metal foil, such as a single layerof an aluminum foil, with no plastic lining layer, as further describedherein.

The containers containing different reagents may be of different colors,or color-coded for easy identification by the user. For example they maybe made of different color material, such as tinted plastic, or may havesome kind of identifying tag on them, such as a color stripe or dot.They may also have a label printed on the side, and/or may have anidentifier such as a 1-D or a 2-D barcode on the sealing layer on thetop, or on the side of the tube. Such a code is useful for identifyingthe composition of the reagents stored within, and/or a batch number forthe preparation thereof, and/or an expiry date. The code may be printedon with, for example, an inkjet or transfer printer.

In one embodiment, the containers 554 containing lyophilized reagents,disposed in the receptacles 550, are 0.3 ml tubes that have been furtherconfigured to have a star-shaped pattern on their respective bottominterior surfaces. This is so that when a fluid has been added to thelyophilized reagents (which are dry in the initial package), a pipettetip can be bottomed out in the tube and still be able to withdraw almostthe entire fluid from the tube. The design of the star-pattern isfurther described elsewhere in U.S. patent application Ser. No.12/178,557, filed on Jul. 23, 2008, and entitled “Reagent Tube”, in thename of Handique et al., which application is incorporated herein byreference. Still other containers used in conjunction with the holderherein may be similarly configured with a start-shaped pattern toincrease pipetting efficiency.

The embodiments of reagent holders 804 are shown configured with a wastechamber 560, having an inlet aperture 562 in the upper side of theconnecting member. Waste chamber 560 is optional and, in embodimentswhere it is present, is configured to receive spent liquid reagents. Inother embodiments, where it is not present, spent liquid reagents can betransferred to and disposed of at a location outside of the holder, suchas, for example, a sample tube that contained the original sample whosecontents are being analyzed.

The embodiments of reagent holders 804 are shown having a pipette sheath570. This is an optional component of the holders described herein. Itmay be permanently or removably affixed to connecting member 510, or maybe formed, e.g., moulded, as a part of a single piece assembly for theholder. Pipette sheath 570 is typically configured to surround the atleast one socket and a tip and lower portion of a pipette tip when thepipette tip is stationed in the at least one socket. In someembodiments, the at least one socket comprises four sockets. In someembodiments the at least one socket comprises two, three, five, or sixsockets. The sheath and sockets are large enough to accommodate avariety of sizes of pipette tips, such as those having volumes as smallas 10 μl to as large as 1 ml.

Pipette sheath 570 typically is configured to have a bottom 576 and awalled portion 578 disposed between the bottom and the connectingmember. Pipette sheath 570 may additionally and optionally have one ormore cut-out portions 572 in the wall 578, or in the bottom 576. Inembodiments of the reagent holder having a pipette sheath, a purpose ofthe sheath is to catch drips from used pipette tips, and thereby toprevent cross-sample contamination, from use of one holder to another ina similar location, and/or to any supporting rack in which the holder issituated. Typically, then, the bottom 576 is solid and bowl-shaped(concave) so that drips are retained within it. An embodiment having nopipette sheath, could utilize, e.g., a drip tray or a drainage outlet,suitably placed beneath pipette tips located in the one or more sockets,for the same purpose and located under or in the bottom of the rack, asdescribed herein.

Process tube 520 (sometimes referred to as a lysis tube) can also be asnap-in tube, rather than being part of an integrated piece. Processtube 520 is typically used for various mixing and reacting processesthat occur during sample preparation. For example, cell lysis can occurin process tube 520, as can extraction of nucleic acids, such as DNA orRNA of a patient, or DNA or RNA of a pathogen. Process tube 520 is thenadvantageously positioned in a location that minimizes, overall, pipettehead moving operations involved with transferring liquids to processtube 520. Process tube 520 is also located in the holder in such aposition that, when the holder is inserted in a rack as furtherdescribed herein, the process tube is exposed and accessible to a heaterand separator, as further described herein. The process tube istypically configured to accept a pipette tip during multiple pipettingoperations.

The process tube also may have a low binding surface, and allowsmagnetic beads to slide up and down the inside wall easily withoutsticking to it. Moreover, it has a hydrophobic surface coating enablinglow stiction of fluid and hence low binding of nucleic acids and othermolecules.

Some of the reagents contained in the holder are provided as liquids,and others may be provided as solids from which a solution isre-generated, in situ, by adding liquid from a pipette tip. In someembodiments, a different type of container or tube is used to storeliquids from those that store the solids.

Reagent tubes 540 are typically configured to hold liquid reagents, oneper tube. For example, in reagent holder embodiment 501, three reagenttubes are shown, containing respectively wash buffer, release buffer,and neutralization buffer, each of which is used in a sample preparationprotocol, carried out with multiple pipetting operations controlled by,e.g., a pipette head as further described herein.

Reagent tubes 540 that hold liquids or liquid reagents can be sealedwith a laminate structure 598. The laminate structure typically has aheat seal layer, a plastic layer such as a layer of polypropylene, and alayer of metal such as aluminum foil, wherein the heat seal layer isadjacent the one or more reagent tubes. The additional plastic film thatis used in a laminate for receptacles that contain liquid reagents istypically to prevent liquid from contacting the aluminum.

Two embodiments of a laminate structure, differing in their layerstructures, are shown in FIG. 5. In both embodiments, the heat seallayer 602, for example made of a laquer or other such polymer with a lowmelting point, is at the bottom, adjacent to the top of the holder, whenso applied. The plastic layer 604 is typically on top of the heat seallayer, and is typically made of polypropylene, having a thickness in therange 10-50 microns. The metal layer 608 is typically on top of theplastic layer and, in one embodiment, may be a layer of Al foil bondedto the plastic layer with a layer of adhesive 606, as in panel A of FIG.5, or, in another embodiment, may be a layer of metal that is evaporatedor sputtered into place directly on to the plastic layer (panel B ofFIG. 5). Exemplary thicknesses for the respective layers are shown inFIG. 5, where it is to be understood that variations of up to a factorof 2 in thickness are consistent with the technology herein. Inparticular, the aluminum foil is 0.1-15 microns thick, and the polymerlayer is 15-25 microns thick in one embodiment. In another embodiment,the aluminum is 0.1-1 microns thick, and the polymer layer is 25-30microns thick.

The laminates deployed herein make longer term storage of reagentseasier because the holder includes both sealed lyophilized reagents andliquids sealed in close proximity, which is normally hard to achieve.

In one embodiment, the tops of the reagent tubes have beveled edges sothat when an aluminum foil is heat bonded to the top, the plastic meltdoes not extend beyond the rim of the tube. This is advantageousbecause, if the plastic melt reduces the inner diameter of the tube, itwill cause interference with the pipette tip during operation. In otherembodiments, a raised flat portion 599 on holders 804 facilitatesapplication and removal of laminate 598. Raised surface 599, on theupper side of the connecting member, and surrounding the inlet aperturesto the reagent tubes and, optionally, the waste chamber, is an optionalfeature of the holder.

The manner in which liquid is pipetted out is such that a pipette tippiercing through the foil rips through without creating a seal aroundthe pipette tip, as illustrated in FIG. 6. Such a seal around the tipduring pipetting would be disadvantageous because a certain amount ofair flow is desirable for the pipetting operation. In this instance, aseal is not created because the laminate structure causes the piercedfoil to stay in the position initially adopted when it is pierced. Theupper five panels in FIG. 6 illustrate, in sequence, the pipetting of areagent 707 (which may be corrosive to direct contact with Aluminum) outfrom a reagent tube 709 sealed with a laminate 598 as further describedherein. At A, the pipette tip is positioned approximately centrallyabove the reagent tube that contains reagent 707. At B, the pipette tip705 is lowered, usually controllably lowered, into the reagent tube, andin so doing pierces the laminate 598. The exploded view of this areashows the edge of the pierced laminate to be in contact with the pipettetip at the widest portion at which it penetrates the reagent tube. At C,the pipette tip is withdrawn slightly, maintaining the tip within thebulk of the reagent 707. The exploded view shows that the pierced foilhas retained the configuration that it adopted when it was pierced andthe pipette tip descended to its deepest position within the reagenttube. At D, the pipette tip sucks up reagent 707, possibly altering itsheight (without bottoming out) as more reagent is removed from the tube.At E, the pipette tip is removed entirely from the reagent tube.

The reagent holder of embodiments 804 has a connecting member 510 thatis configured so that the at least one socket, the one or morereceptacles, and the respective apertures of the process tube, and thetwo or more reagent tubes, are all arranged linearly with respect to oneanother (i.e., their midpoints lie on the same axis). However, theholders herein are not limited to particular configurations ofreceptacles, process tube, sockets, reagent tubes, and waste chamber ifpresent. For example, a holder may be made shorter, if some aperturesare staggered with respect to one another and occupy ‘off-axis’positions. The various receptacles, etc., also do not need to occupypositions with respect to one another that are the same as those shownin FIG. 3A, 3B, or 4. Thus, in FIGS. 3A and 3B, the process tube is onone end of the connecting member, and the pipette sheath is at the otherend, adjacent to, in an interior position, a waste chamber and two ormore reagent tubes. Still other dispositions are possible, such asmounting the process tube on one end of the holder, mounting the processtube adjacent the pipette tips and pipette tip sheath, and mounting thewaste tube adjacent the process tube (see FIG. 4). It would beunderstood that alternative configurations of the various parts of theholder give rise only to variations of form and can be accommodatedwithin other variations of the apparatus as described, including but notlimited to alternative instruction sets for a liquid dispensing pipettehead, heater assembly, and magnetic separator, as further describedherein. Each such configuration of the reagent holder can beaccommodated by a corresponding variation in form of the rack describedherein that receives one or more such holders.

In some embodiments, the holder comprises a registration member such asa mechanical key. Typically such a key is part of the connecting member510. A mechanical key ensures that the holder is accepted by acomplementary member in, for example, a supporting rack as describedherein or a receiving bay of an apparatus that controls pipettingoperations on reagents in the holder. Thus, embodiment 501 has amechanical key 592 that comprises a pair of rectangular-shaped cut-outson one end of the connecting member. This feature as shown additionallyprovides for a tab by which a user may gain a suitable purchase wheninserting and removing the holder into a rack or another apparatus.Embodiment 501 also has a mechanical key 590 at the other end ofconnecting member 510. Key 590 is an angled cutout that eases insertionof the holder into a rack, as well as ensures a good registrationtherein when abutting a complementary angled cut out in a recessed areaconfigured to receive the holder.

In some embodiments, not shown in FIG. 3A, 3B, or 4, the holder furthercomprises an identifier affixed to the connecting member. The identifiermay be a label, such as a writable label, a bar-code, a 2-dimensionalbar-code, or an RFID tag. The identifier can be, e.g., for the purposeof revealing quickly what combination of reagents is present in theholder and, thus, for what type of sample preparation protocol it isintended. The identifier may also indicate the batch from which theholder was made, for quality control or record-keeping purposes. Theidentifier may also permit a user to match a particular holder with aparticular sample.

It should also be considered consistent with the description herein thata holder additionally can be configured to accept a sample, such as in asample tube. Thus, in embodiments described elsewhere herein, a rackaccepts a number of sample tubes and a number of corresponding holdersin such a manner that the sample tubes and holders can be separately andindependently loaded from one another. Nevertheless, in otherembodiments, a holder can be configured to also accept a sample, forexample in a sample tube. And thus, a complementary rack is configuredto accept a number of holders, wherein each holder has a sample as wellas reagents and other items. In such an embodiment, the holder isconfigured so that the sample in a suitably marked tube or container isaccessible to a sample identification verifier.

A reagent holder for use with a rack as described herein is typicallymade of a plastic such as polypropylene. The plastic is such that it hassome flexibility to facilitate placement into a rack, as furtherdescribed herein. The plastic is typically sufficiently rigid, however,so that the holder will not significantly sag or flex under its ownweight and will not easily deform during routine handling and transportor pipetting operations as further described herein, and thus will notpermit reagents to leak out from it.

The holder is typically such that the connecting member, process tube,the two or more reagent tubes, and the waste chamber (if present) aremade from a single piece, made from a material such as polypropylene.

The materials of the various tubes and chambers may be configured tohave at least an interior surface smoothness and surface coating toreduce binding of DNA and other macromolecules thereto. Binding of DNAis unwanted because of the reduced sensitivity that is likely to resultin subsequent detection and analysis of the DNA that is not trapped onthe surface of the holder.

Rack

The apparatus outlined herein, and also described in U.S. patentapplication Ser. No. 12/173,023, filed by ExpressMail on Jul. 14, 2008(and entitled “Integrated Apparatus for Performing Nucleic AcidExtraction and Diagnostic Testing on Multiple Biological Samples”, inthe name of Williams, et al.), incorporated by reference herein, isconfigured to carry out various liquid transfer operations on samplesand various reagents, in parallel. The samples and various reagents aretypically held in one or more removable racks 970, positioned in theapparatus (such as one shown in FIG. 1, 2A, or 2B), while the variousliquid transfer operations are carried out. Optionally, the operationscan be carried out on the reagents, stored in holders located directlyin the apparatus, without use of a removable rack.

The racks for use herein are typically configured to be insertable into,and removable from, a diagnostic or preparatory apparatus as furtherdescribed herein (e.g., in connection with FIGS. 1, 2A and 2B), each ofthe racks being further configured to receive a plurality of reagentholders, and to receive a plurality of sample tubes, wherein the reagentholders are in one-to-one correspondence with the sample tubes, andwherein the reagent holders each contain sufficient reagents to extractpolynucleotides from a sample and to place the polynucleotides into aPCR-ready form. Exemplary racks are further described in U.S. patentapplication Ser. No. 12/178,584, filed Jul. 23, 2008, to Duffy et al.,incorporated herein by reference in its entirety.

Two perspective views of an exemplary rack 800, configured to accept 12sample tubes and 12 corresponding reagent holders, in 12 lanes, areshown in FIG. 7. A lane, as used herein in the context of a rack, is adedicated region of the rack designed to receive a sample tube andcorresponding reagent holder. A perspective view of the same exemplaryrack, in conjunction with a heater unit, as further described herein, isshown in FIG. 8. The lanes of the rack described herein are designed tohave sufficient depth and width to accommodate the various reagenttubes, receptacles, process tube, and pipette sheath of a given reagentholder as described elsewhere herein, and to position the process tubein communication with a heater/separator unit.

A rack may accept 2, 4, 6, 8, 10, 12, 16, or 20 samples such as insample tubes 802, and a corresponding number of reagent holders 804.Thus the embodiment of FIG. 8, configured to receive 12 samples insample tubes 802, and 12 corresponding reagent holders 804, isexemplary.

Rack 800 is shown with a handle 806, having optionally a hand-grip 808,to facilitate transport, and removal from the apparatus. Rack 800 isalso shown with positioning feet 811 that can help stabilize the rackduring loading and when resting on, e.g., a bench-top, outside of theapparatus. Rack 800 is also shown as having a structural member 810,typically made of steel, that provides strength and rigidity for therack, and also ensures that the rack fits tightly into an appropriatelyconfigured receiving area of the apparatus. Rack 800 is also shown ashaving a body 812 configured with a number of slots that accept thereagent holders.

As described elsewhere herein, the holders each comprise a process tubein which reactions, e.g., between reagents and sample, take place,typically with some heating, or cyclical heating and cooling. Thelocation of the reagent holders in the rack typically ensures that theprocess tubes are effectively located in proximity to the heater units,as shown in FIG. 8.

Heater Assembly & Magnetic Separator

The racks as described herein are configured such that the reagentholders placed in the racks are positioned so that the process tubes inthe holders are heated by a dedicated heating assembly 977, as may besituated in an apparatus for carrying out sample preparation andanalysis on multiple samples in parallel, such as shown in FIG. 1, 2A or2B. Typically such a heater assembly comprises one or more independentlycontrollable heater units 1010, each of which comprises a heat blockconfigured to heat a process tube in a reagent holder situated in therack, as further described herein. In one embodiment, a heat element isa power resistor. The right hand panel of FIG. 8 shows how holdersloaded in a rack can be positioned in close proximity to such adedicated heating unit. The heating unit is configured to heat theprocess tube in each of one or more reagent holders positioned in therack, without unduly heating other portions of the rack, or othercontainers associated with the reagent holders.

Yet additionally, the holders herein are configured so that each processtube is in close enough proximity to a magnetic assembly that separationof magnetic particles from reagents in solution in the process tubes canbe accomplished. An exemplary magnetic separator is configured to moveone or more magnets relative to the one or more process tubes.Typically, the magnet is mounted in such a way that it can be moved inproximity to the process tubes, either in an automated fashion such asunder control of a processor, or manually. The magnet can be made ofneodymium (e.g., from K&J Magnetics, Inc.) and can have a magneticstrength of 5,000-15,000 Gauss (Brmax). The poles of the magnets can bearranged such that one pole faces the heat blocks and the other facesaway from the heat blocks.

Advantageously, the heater assembly and magnetic separator operatetogether to permit successive heating and separation operations to beperformed on liquid materials in the one or more process tubes withouttransporting either the liquid materials or the process tubes todifferent locations to perform either heating or separation. Anexemplary heater assembly and magnetic separator are further describedin U.S. provisional Patent Application Ser. No. 60/959,437, filed Jul.13, 2008, and U.S. patent application Ser. No. 12/173,023, filed Jul.14, 2008, entitled “Integrated Apparatus for Performing Nucleic AcidExtraction and Diagnostic Testing on Multiple Biological Samples”, inthe name of Williams, et al., and Ser. No. 12/178,586, entitled“Integrated Heater and Magnetic Separator”, in the name of Handique,filed on Jul. 23, 2008, all of which are incorporated herein byreference in their entirety.

The heater assembly and magnetic separator are also configured tooperate in conjunction with the liquid dispenser further describedherein so that, when appropriate quantities of liquid reagents and/orsample have been dispensed into the process tube adjacent the heater andseparator, the heater and separator are controllably activated toaccomplish the required heating and/or separating.

Pipetting Operations

Basic pipetting operations, such as may be accomplished with theautomated pipetting apparatus described herein, are now described, asfollows. FIG. 9 has a number of panels, A-G, each representing, insequence, a stage in an exemplary pipetting operation, such as may becarried out with a pipette head as described further herein and aprocess tube, as described elsewhere herein. At A, a pipette tip 2210,containing a liquid 2211 (such as a buffer solution), is positioneddirectly or approximately above the center of reagent tube 2200. Thetube contains a number of lyophilized pellets 2212, and is sealed by alayer 2214, such as of foil. The foil may be heat-sealed on to the topof the tube. Although a laminate layer, as further described herein, canbe placed on the reagent tube, typically a layer of aluminum foil isadequate, where the tube contents are solid, e.g., lyophilized,reagents. In some embodiments, the top of the reagent tube has chamferedges to reduce expansion of the top rim of the tube during heat sealingof a foil on the top of the tube.

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis using the apparatus as further described herein,can include one or more of the following steps: contacting a neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides (in some embodiments, the PCRreagent mixture can further include a positive control plasmid and afluorogenic hybridization probe selective for at least a portion of theplasmid); in some embodiments, the PCR reagent mixture can be in theform of one or more lyophilized pellets, as stored in a receptacle on aholder, and the method can further include reconstituting the PCR pelletwith liquid to create a PCR reagent mixture solution. Various, such asone or more, of the liquid transfer operations associated with theforegoing steps can be accomplished by one or more pipette heads on anautomated pipetting apparatus that comprises a liquid dispenser, asfurther described herein.

The automated liquid dispenser can be further configured to dispense asolution (e.g., of a prepared sample, various PCR reagents, anddetection tags) into a microfluidic cartridge. Thus, the liquiddispenser is configured to travel from a first set of positions abovereagent holders having various containers that hold reagents, etc., to asecond set of positions above the inlets of a microfluidic cartridge.The second set of positions is depicted schematically in FIG. 10, inside cross-sectional view. The travel of the liquid dispenser betweenthe first set of positions and the second set of positions can beaccomplished by motions in combinations of two orthogonal directions ina horizontal plane, for example, along supporting structures as furtherdescribed herein, and under control of a microprocessor. Although notapparent from FIG. 10, it is consistent with the depiction thatmultiple, e.g., 4, pipette tips are dispensing fluid into differentinlets of microfluidic cartridge 994 at any time. Liquid dispenser 976has attached a pipette tip 1807 that is positioned so that its tip isinserted into an inlet 202 of a microfluidic cartridge 994. Thecartridge is situated in a receiving bay 992. An optional cover 310 isconfigured to shut out ambient light from the remainder of cartridge994, where, e.g., a target polynucleotide is detected after PCR, so thatdetector 300 can be as effective as possible. Suitable detectors aredescribed in, e.g., U.S. patent application Ser. No. 12/218,498, filedJul. 14, 2008, and incorporated herein by reference in its entirety.Although it is to be understood that the liquid dispenser herein istypically configured for use with a microfluidic cartridge, it canequally be configured to deliver appropriate quantities of preparedpolynucleotide in solution to other locations at which suchpolynucleotides can be amplified and detected.

Liquid Dispenser

The liquid dispenser, as further described herein, can be configured tocarry out pipetting operations in parallel on samples and solutionsstored in one or more holders, and in one or more sample tubes, in arack, as described elsewhere herein. It would be understood, however,that the operation, design, and function of the liquid dispenser is notdependent upon the locations of the samples and various solutions, butthat the liquid dispenser could perform similarly in connection withpipetting solutions disposed in other types of receptacles. Thus, aliquid dispenser, as described herein, is an assembly of components thattogether cooperate to carry out such pipetting operations on solutions.The liquid dispenser thus, typically, can pick up and drop off pipettetips as needed, as well as aspirate quantities of liquid up into, anddeposit out those quantities of liquid from, such pipette tips. Themotions and operation of the liquid dispenser is typically controlled bya processor such that pipetting operations can be automated.

Advantageously, the liquid dispenser can be configured so that thepumps, sensors (e.g., for pipette tip presence detection, and forcesensing during pipetting), sample identification verifier, and otheritems, move with it, and therefore minimize the number of control linesthat move across the instrument during use, and also reduces thelikelihood that such control lines will become tangled during motion ofthe liquid dispenser, as would be the case where pipette dispense headsare the only items undergoing motion, and remain in communication withother components that are fixed at various points within a preparatoryor diagnostic apparatus. In such apparatus, where only e.g., dispenseheads undergo motion, the need to be able to move freely in threedegrees of freedom becomes severely constrained by the need to move anumber of cables independently of one another.

Advantageously, as further described herein, also, the dispenser can beconfigured to align pipette tips, e.g., with cartridge inlet holes,using a motorized alignment plate. Additionally, as also describedelsewhere herein, the dispenser can be configured with a scanner thatreads information from, e.g., a sample.

FIG. 11 shows, schematically, components of a liquid dispenser 4000 asfurther described herein. The layout of the components in FIG. 11 is forconvenience only, and one of skill in the art would appreciate thatother arrangements are possible, depending upon environment and otherfactors. A support 4001 has three dispense heads 4002 mounted to it.Other numbers of dispense heads, such as 1, 2, 4, 5, 6, 8, and 10, areconsistent therewith. The dispense heads are configured to acceptpipette tips 4003-1 (shown detached from its head), and 4003-2, shownmounted on the head. The support 4001 is movably attached via aconnecting member to a mount 4017. The relative position of the supportand the mount, in the z-direction as shown, can be controlled by Z-motor4013, which is electrically coupled via connection 4014 to the support4001. Z-motor receives instructions from a processor (not shown) via aconnection 4019. In the embodiment shown, Z-motor is able to control therelative position of support 4001 and mount 4017 by moving support 4001.In other embodiments, Z-motor 4013 is coupled to mount 4017 and achievessimilar relative motion of mount and support. Such relative motion canbe accomplished by any suitable mechanical movement device, such asgearing, or a rack and pinion assembly, or a lead screw, the details ofwhich are not shown in FIG. 11.

Also included within the liquid dispenser 4000 is a sensor 4004configured to sense when vertical motion of the support or mount isobstructed, and to provide a suitable signal, e.g., via an electricalconnection 4020, directly to a processor (not shown), or indirectly (notshown) via printed circuit board 4008. Thus sensor 4004 can be mountedon support 4001, as shown, or on mount 4017, depending on matters ofdesign choice.

Optionally included within the liquid dispenser 4000 is a scanner 4015,connected to, e.g., support 4001 (or, alternatively, to mount 4017) viaa connector, such as a mechanical attachment, 4016. Scanner 4015 can beconfigured to read, e.g., sample and patient information, from one ormore of a sample tube, reagent holder, or microfluidic cartridge, asfurther described elsewhere herein. Scanner 4015 can be electricallyconnected directly (not shown) to a processor, or indirectly via printedcircuit board 4008.

A valve 4005 is associated with each dispense head 4002, and serve tocontrol operation of each dispense head such as by, for example,controlling when to reduce pressure, thereby causing a suckingoperation, or to increase pressure, thereby causing a dispenseoperation. Each valve 4005 is connected to (including being in fluidcommunication with) manifold 4007 via a connecting tube 4006.

Manifold 4007 is connected to pump 4012 via an air-line 4011, and tovalves 4005 via connecting tubes 4006. Manifold 4007 contains a numberof independently controllable valves that selectably divert air frompump 4012 to various of valves 4005, and therefore to correspondingdispense heads 4002. In FIG. 11, a way to accomplish this is shownschematically: line 4011 is split into three separate lines each ofwhich connects to one of lines 4006. In embodiments that servicedifferent numbers of dispense heads, such as 4 heads, line 4011 issimilarly split into 4 corresponding lines.

Manifold 4007 is also typically connected to pump 4012 via a second line4020 that is configured to permit equilibriation of air between manifoldand pump. Line 4020 connects to a vent 4021 on the manifold, and is alsocontrolled by a valve 4022.

Operation of manifold 4007 is typically controlled by printed circuitboard (PCB) 4008 to which it is connected via an electrical connection4009. PCB 4008 additionally can receive electrical input from connection4010. Thus, the suck and dispense operations can be preciselycontrolled, by signals from the PCB, so that accurate volumetric controlis achieved. In some embodiments, calibration of the liquid dispenser isrequired so that the amount of time to force or to suck air that isrequired to dispense or aspirate a desired volume of liquid is known.Thus, the time between, e.g., a valve opening and valve closing, ascontrolled by signals, is known and can be incorporated into the controlsoftware.

Pump 4012 typically also comprises a motor (not shown) controlling itsaction, e.g., motion of a plunger, which receives electrical signals asinput, and an air supply (not shown).

FIGS. 12-21 (inclusive) show various views of an exemplary liquiddispenser, now various components of which are further described herein.It would be understood by one of ordinary skill in the art that suchcomponents, their relative configuration, number, and orientation, areexemplary, and that the degrees of freedom of motion, and accuracy ofpositioning and dispensing, consistent with the description herein maybe achieved by other such configurations. For example, where one or moremounts are shown, other embodiments may have different numbers ofmounts.

A perspective side view of an exemplary liquid dispense head is shown inFIG. 12. The following items relate to control of movement of the liquiddispenser, and the housing of the liquid dispenser, are visible. Controlbelts 2120 and 2121 house electrical cables, are disposed orthogonallyto one another, and permit motion of the liquid dispenser in twoorthogonal directions: in a horizontal and a vertical plane. Controlbelts 2106 and 2107 hold further electrical cables, and are disposed topermit motion in a horizontal plane, orthogonal to belt 2121. Belts2106, 2107, 2120, and 2121 permit easy motion of the liquid dispenserwithout entangling various electrical cables because the belts guide andhouse the cables while the dispenser is in motion. Electrical cable 2125supplies control signals to assembly 2144, which houses electricalcircuitry to control operation of manifold 1802 and a pump 2141 of theliquid dispenser. Manifold 1802, attached to pipette heads and otheritems as described herein, is thereby capable of moving up and down(z-axis), as well as in two horizontal directions. Electrical cable 1702supplies control signals to assembly 2101, which is coupled to a motorfor accomplishing vertical motion, and thereby permits such motion to becontrolled. Assembly 1700 is a housing that holds the motor and thesliding head and is attached to one or more mounting plates 2104, 2142,which at least one of which is attached to a gantry 2108. A mountingassembly 2140 connects the liquid dispenser to the assembly 1700 thatcontrols vertical motion. Mounting assembly 2140 can further comprise anair displacement/plunger pump for directing air to the dispense head. Afurther mounting 2129 serves as a shield for the pipette dispense heads.

The gantry 2108 comprises a horizontal rail 2102 to provide movement inthe x-direction, controlled by controller 2109, which receiveselectrical input from cables (not shown). Also not shown is anorthogonally disposed rail to provide movement in the y-direction of therail and the attached assemblies. The gantry permits, overall, threedegrees of translational freedom of the liquid dispenser. (Furtherembodiments, not herein described, can comprise a gantry having fewerthan three degrees of translational freedom.) A suitable gantrycomprises three axes of belt-driven slides actuated by encoded steppermotors. The gantry slides can be mounted on a framework of structuralangle aluminum or other equivalent material, particularly a metal ormetal alloy. Slides aligned in x- and y-directions (directed out of andin the plane of FIG. 12 respectively) facilitate motion of the dispenseracross an array of holders, and in a direction along a given holder,respectively. The z-axis of the gantry can be associated with a variableforce sensor which can be configured to control the extent of verticalmotion of the head during tip pick-up and fluid dispensing operations,as further described herein.

Assembly 1700 is shown only as an outer housing; internal parts arefurther shown in FIGS. 13A and 13B. A manifold 1802 is attached to anassembly 2140; the manifold controls suck and dispense operationsperformed by multiple pipette heads (not shown in FIG. 12). Assembly2140 can undergo vertical movement, under suitable control, and is alsofurther illustrated in FIGS. 13A and 13B. A detector 1701 is mountedindirectly to assembly 2140 and therefore can also move in a verticaldirection. Detector 1701 typically permits positive detection of sampletubes, reagent disposables, and microfluidic cartridges. Electricalcable 2126 provides control signals to detector such as a scanner, orread-head 1701. A motor 2130 is a positioned to control motion of astripper plate for stripping pipette tips, as further described herein.Electrical control of stripper motor 2130 can be provided by variouselectrical cables such as 2128 as shown in FIG. 12.

As shown in the various figures, the entire liquid dispenser that movesup and down the z-axis is a self-contained unit having only electricalconnections to a processor or controller, and mechanical connections tothe gantry. The translational motions in three dimensions of the liquiddispenser can be controlled by a microprocessor, such as processor 980.No fluid handling lines are associated with the dispenser. This designenables simplification of assembly of the instrument, minimizescontamination of the instrument and cross-contamination of samplesbetween different instances of operation of the apparatus, increasesefficiency of pumping (minimal dead volume) and enables easy maintenanceand repair of the device. This arrangement also enables easy upgradingof features in the dispensing device, such as individual and independentpump control for each dispenser, individual pipette attachment orremoval, ability to control the pitch of the pipettes, etc.

A suitable liquid dispenser for use with the apparatus herein comprises:one or more sensors (such as for sensing pipette tips, in FIGS. 17A-17C,and as further described herein); a manifold 1802; one or more pumps2141 in fluid communication with the manifold; one or more dispenseheads 1803 in fluid communication with the manifold; and electricalconnections that accept electrical signals from an external controller,wherein the liquid dispenser has no inlet or outlet for fluids, otherthan through the one or more pumps. As described elsewhere herein, theliquid dispenser can be configured to carry out fluid transferoperations on two or more holders simultaneously, such as when operatingunder instructions received from one or more electrical controllers.Other sensors incorporated into the apparatus include: a sensor to sensewhen a pipette tip reaches the bottom of a sample tube (also called anencoder/stall sensor, as further described herein); and sensors thatrestrict motion of the stripper plate so that it moves back and forthbetween two limit switches.

A cross-sectional view of the exemplary liquid dispenser of FIG. 12 isshown in FIGS. 13A and 13B. FIG. 13B shows in close-up a portion(dashed-line box) of FIG. 13A. (Various items visible in FIG. 12, suchas control cables, are omitted from FIGS. 13A and 13B, for clarity.)Liquid dispenser 2100, and ancillary items shown in FIGS. 13A and 13B,are mounted on a gantry (not shown) via a support 2104. The manner ofmounting can be by a supporting member 2110, such as a plate, to whichthe dispenser is attached via a mechanical fastening such as one or morescrews 2111. In the embodiment of FIG. 13A, a lead screw 2112 (shown incross-section) couples the z-motor with the whole z-head and provides amechanism that permits the z-head to move up and down vertically.

Typically, pipette heads 1803 are individually sprung. Shown in FIGS.13A, 13B, for example, a pipette head 1803 can be mounted such that aforce acting upwardly against the head, such as created when a pipettetip attached to the head meets the bottom of a container from whichliquid is being sucked, can be sensed through a relative motion betweenthe head and a force sensor. For example, when a tip attached to pipettehead 1803 forces against a disposable holder in a rack below it, anupward force is transmitted causing head 1803 to torque around pivotpoint 2122, causing set screw 2124 to press against a force sensor. Inturn, the force sensor is in communication with a processor orcontroller on PC board 2120 that controls at least the vertical motionof the liquid dispenser so that, thereby, the processor or controllercan send instructions to arrest the vertical motion of the liquiddispenser upon receiving an appropriate signal from the force sensor. Anexemplary force sensor suitable for use herein is available fromHoneywell. The force sensor mechanism shown in FIGS. 13A and 13B isexemplary and one of many possible mechanisms capable of commanding thehead during up pick-up and fluid dispensing operations. For example, asan alternative to a force sensor, a stall sensor that sensesinterruption in vertical motion of the one or more dispense heads uponcontact with a sample tube or reagent holder may be used. In someembodiments, the stall sensing is performed by the encoder of thez-motor. The encoder is a sensor attached to the motor and it senses anyangular steps performed by the motor. During stalling of the z-head, theencoder senses that the motor has stopped moving even though the motorwas instructed to go beyond the position at which it stalled.Accordingly, as would be understood by one of ordinary skill in the art,the upward motion of the liquid dispenser as described herein is notlimited to the specific mechanism shown in FIGS. 13A and 13B. A lengthof tubing 2131 is attached between the fluidic manifold 1802 and each ofthe pipette attachment nozzles.

FIGS. 14A-14C show an exemplary liquid dispenser in close-up, inperspective (FIG. 14A), side (FIG. 14B, enlarged to show a portion ofwhat is visible in the view of FIG. 14A), and front (FIG. 14C) views.The liquid dispenser comprises a number of individually sprung heads1803, wherein each head is configured to accept a pipette tip, such asfrom the one or more pipette tips in a holder as elsewhere describedherein. Thus the spacing of the heads is calculated to be the same asthe spacing of the holders in a rack, as further described herein. Therightmost head is shown with a pipette tip 1807 attached to it, visiblein FIGS. 14A and 14C. The liquid dispenser can be further configuredsuch that no two heads accept pipette tips from the same holder. Theliquid dispenser can be used with, or be adapted to be used with pipettetips that have volumes as small as 10 μl to as large as 1 ml.

FIGS. 14A-C depict, for example, a “4-up” automated pipetting apparatushaving four individually sprung heads 1803, but it is to be understoodthat the dispenser is not limited to this number. For example, othernumbers include 2, 3, 5, 6, 8, 10, or 12. Furthermore, the individuallysprung heads 1803 are shown arranged in a line in FIG. 14A, but may beconfigured in other arrangements, such as an array, or a circle.

The liquid dispenser can further comprise computer-controlled,motorized, pump 1800 connected to distribution manifold 1802 withrelated computer-controlled valving. The distribution manifold typicallytravels with the dispense head, rather than being positioned at a fixedlocation away from the dispense head while the dispense head moves fromone pipetting location to another. Computer-control can be accomplishedvia a control board 1809, shown in the embodiment of FIGS. 14A-14Cmounted on the front of the liquid dispenser. It would be understoodthat, in other embodiments, the control board could be mountedelsewhere, including at locations other than on the liquid dispenser ifit is desired to run electric cables to the dispenser.

Also shown in FIGS. 14A-14C are a number of connectors 1811 for tubingthat extends from the pump to the fluidic manifold. A mechanicalstructure 1821 maintains the four pipette nozzles at a fixed distanceand location relative to the z-head.

The liquid dispenser is typically configured to aspirate or dispensefluid in connection with analysis or preparation of solutions of two ormore samples. However, that is not to say that any of the featuresdescribed herein could not also be applied in a device that operates ona single sample. The liquid dispenser is also configured to dispenseliquid into a microfluidic cartridge. Typically, the liquid dispenser isconfigured to accept or dispense, in a single operation, an amount of1.0 ml of fluid or less, such as an amount of fluid in the range 10 nl-1ml.

The liquid dispenser is configured such that pump 1800 pumps air in andout of the distribution manifold. The pump can have an air supply andcan be as simple in construction as having a plunger that moves back andforward compresses/expands air volume, under control of a motor, whoseoperation is in turn controlled by electrical signals from a processor.Air can be supplied to pump 1800 and is typically under pressure, suchas at 0.1-10 psi. Thus the air supply may ultimately be provided by acompressed air cylinder, located outside of the apparatus. Typically thepump communicates with the manifold via two airways. A first airway,directs pressurized air from the pump to the manifold. A second airwaycan be for the purpose of equilibriating, where required, betweenvarious pipette operations, and connects with a vent on the manifold.When the pump draws air in, it is typical to close off the vents andvalves in the manifold.

Further shown in FIG. 14A is a vent 1819, usually equipped with a filter(so that any airborne particles are trapped). Vent 1819 is usuallyclosed unless it is necessary to prime the pump (such as whenequilibriating the airways).

Fluid distribution manifold 1802, of which an exemplary embodiment isshown in FIG. 13, can comprise a number of valves, such as solenoidvalves 1801, as are available from, e.g., the Lee Co., configured tocontrol the flow of air through the pipette tips. Construction anddesign of such a manifold is within the capability of one skilled in theart. In an exemplary embodiment, there are two valves for each pipette,and one additional valve to vent the pump. Thus, for a liquid dispenserhaving four pipette heads, there are nine valves. In another embodimentthere is only one valve for each pipette, and one additional valve tovent the pump. However, the distribution manifold is not limited tocomprising exactly nine or exactly five solenoid valves.

The distribution manifold comprises a microfluidic network 1829 thatdistributes air evenly amongst the one or more valves that individuallyregulate air flow to the dispense heads. Thus, by controlling flow ofair through the manifold and various valves, pressure above the pipetteheads 1803 can be varied so that liquid is drawn up into or expelledfrom a pipette tip attached to the respective pipette heads. In this wayit is not necessary to supply compressed air via an air hose to theliquid dispenser. Neither is it necessary to provide liquid lines to thedispense head. Furthermore, no liquid reagents or liquid samples fromthe holders enter any part of the liquid dispenser, including themanifold. The volume of liquid drawn into the pipette is less than themaximum volume of the pipette, and therefore overflows are avoided. Thisaspect reduces complications that would arise if air bubbles areintroduced into samples or liquid reagents. An exemplary configurationof a microfluidic network in a distribution manifold is shown in dashedlines in FIG. 15. A microfluidic network is advantageous because it islightweight and compact, and easy to manufacture.

Pipette Tip Stripper

The liquid dispenser can also operate in conjunction with a motorizedplate configured to strip the pipettes and align the pipettes duringdispensing of fluid from multiple pipette tips simultaneously, e.g.,into a microfluidic cartridge, as further described herein. Such adevice is found to be important because the tolerances for incorrectpositioning of a pipette tip are very fine.

FIGS. 16A and 16B show operation of an exemplary device for strippingpipette tips from a liquid dispenser as further described herein. FIG.16A is a front plan view of an embodiment of a dispense head, mounted ona gantry, as also shown in FIG. 12. A structure 1828 that holds 4infra-red detectors for pipette sensing is shown. On the opposite sideof structure 1828 (not shown) there are a number of infra-red LED's thatsend infra-red light towards the infra-red detectors. Typically thenumber of such LED's is the same as the number of detectors, in thiscase four. In the presence of pipette tips, an infra-red detector sees aloss of infra-red signal intensity. Also shown in FIG. 16A are sampletubes 1830, configured to accept pipette tips during various pipettingoperations.

FIG. 16B shows a perspective view of a pipette stripper. The pipettetips 1807 are aligned, all at the same pitch, above respective sockets(e.g., over a pipette tip sheath) in a holder. A metal plate 1833 havingone elongated hole 1835 per pipette tip lies over the sockets. Metalplate 1833 serves to play both alignment and stripping roles. Hole 1835is configured so that it is wide enough to accommodate a pipette tip,but also has an angled elongated portion that can grip a pipette tip.Electrical connections 1839 to motor 1831, that controls sidewaysmovement of plate 1833, are shown.

In a stripping role, as illustrated in FIGS. 17A-17B, the pipette tips(attached to the dispense head) are inserted part way down into thesheath through the elongated holes, for example under control of theliquid dispenser herein, and the metal plate is moved sideways, such asunder control of a motor 1831, in such a manner that the pipette tipsare clamped by the elongated portion of the holes. When the liquiddispenser is moved up, the pipette tips become detached from theirrespective heads. When the metal plate is subsequently moved back to itsinitial position, the pipette tips remain in place in their respectivesockets.

In an aligning role, shown in FIGS. 17A-17B, similar operations areperformed except that the metal plate is moved sideways sufficiently tocontact each pipette tip but not so far as to clamp any tip. The motionof the plate is such that the tips become aligned with respect to oneanother. FIG. 17C shows an outcome of aligning four pipette tips; thetips are positioned over four respective inlets 2303 of a microfluidiccartridge 2301, so that liquid can be loaded into the cartridge byinterfacing the pipette tips with dedicated inlet holes, such as conicalinlet holes, on the cartridge.

In certain embodiments, the liquid dispenser can also comprise one ormore sensors 2001 (e.g., infra-red sensors) each of which detects thepresence of a pipette tip 2005 in position beneath the dispense heads,such as in one or more holders in a rack as further described herein.This is important to ensure that the processor knows affirmatively thata pipette tip is present or missing. Since a pipette tip is picked up byapplication of mechanical force of a head against the pipette, and isalso dispensed using mechanical motion of a stripper plate, sensing apipette tip helps prevent mechanical errors such as having a headdescend too far and become damaged. The embodiment in FIG. 18 shows 4infrared sensors 2001 for detecting the presence of pipettes attached tothe 4 pipette heads.

Such sensors can be mounted in close proximity to the pipette tipstripper described elsewhere herein. In FIG. 18, for example, aninfra-red sensor 2001 can have an infra-red emitter 2003 (not shown, buton the reverse side of plate 2000) placed opposed to it, so that thepresence of disposable pipette tip 1807 obstructs the line of sightbetween the emitter and the detector, thus enabling determination of thepresence or absence of the pipette tip. The disposal pipettes areconfigured perpendicular to pipette stripper-alignment plate 1833 asfurther described herein.

The embodiment shown in FIG. 18 has a stripper/alignment plate 1833 thatis not flat but undulating. In other embodiments, the stripper plate canbe flat, grooved, or have other shapes, such as having a wedge-shapedcross-section.

Sample Identification Verifier

Another aspect of the apparatus relates to a sample identificationverifier configured to check the identity of each of the number ofsamples, and typically mounted on one face of the liquid dispenser, theface and location on the face being determined by other geometricfeatures of the apparatus and its various components, as may beroutinely optimized by those of skill in the art. Such sampleidentification verifiers can be optical character readers, bar codereaders, or radio frequency tag readers, or other suitable readers, asavailable to one of ordinary skill in the art. A sample identificationverifier can be mounted on the gantry to which the liquid dispenser ismounted, or attached to the liquid dispenser so that it moves in concertwith the liquid dispenser. Alternatively, the sample identificationverifier can be separately mounted and can move independently of theliquid dispenser.

In FIGS. 19 and 20, for example, sample identification verifier 1701 isa bar-code reader attached to the liquid dispenser. In FIG. 19, thedispense head is positioned over several reagent holders 804, mounted ina rack in a diagnostic apparatus. The sample identification verifier issimilarly positioned, such that it can read labels situated on the topsof the various holders 804. Aperture 1703 determines the field of viewof the verifier.

In the view of FIG. 20, the verifier is positioned to read identifyingmarks on sample tubes 802. The field of view 1705 of barcode scanner1701 is non-linear, enabling it to detect light reflected by mirror 1705from, e.g., a the barcoded clinical sample tube 802, in disposable rack812. The barcode scanner reads the barcode on the clinical sample tubethus identifying the presence and specifics of the sample tube. Becauseof use of a mirror, the scanner is configured either to read a bar-code,or a 2-D barcode, printed in mirror image form (that is thus reflectedinto normal form by the mirror), or to read a mirror image of a normalbar-code and to convert the mirror image to unreflected form via acomputer algorithm.

In FIG. 21, the sample identification verifier is positioned to readindicia from a microfluidic cartridge 994, located in a receiving bay992.

The verifier is typically mounted so that freedom of motion along thez-axis permits it to be readily positioned to read the sample tube,holder, and cartridge barcodes.

Sample identification verifier is configured to communicate details oflabels that it has detected or read to a processor 980 or controller inthe apparatus, thereby permitting sample identifying information to beassociated with diagnostic results and other information relating tosample preparation, and extraction and amplification of nucleic acidtherein.

Processor and Control

Control of automated motions of the liquid dispenser of the automatedpipetting apparatus is via a suitably configured processor. Theprocessor has been configured to execute instructions that delivercontrol signals to the various motors, and to receive signals from thevarious sensors, within the automated pipetting apparatus. Design andmanufacture of such a processor is within the capability of one ofordinary skill in the art of laboratory automation systems, or apparatuscontrol systems. The instructions executed by the processor can,similarly, be designed and implemented by one of ordinary skill in theart of computer programming. The instructions can take into accountdesired protocols of varying natures, depending on numbers of samples,locations of samples, and nature of target nucleotides, and causemotions of the liquid dispense head. The instructions can also take intoaccount signals received from one or more sensors, in order to determinewhich of one or more next steps to execute, or whether to execute suchsteps at all or to instead, issue an error notification. Theinstructions may provide to a user a menu of pre-determined protocols tochoose from and to execute, or may permit a user to design a newprotocol, or modify an existing one.

Microfluidic Cartridge

As described elsewhere herein, the liquid dispenser can be configured todeliver quantities of solution containing one or more polynucleotide(s)in a form suitable for amplification to a microfluidic cartridge.Typically, such delivery occurs for multiple quantities of solution inparallel. A microfluidic cartridge compatible with such a processtypically has a number of inlets, corresponding to a practical number ofsamples that are to be processed in parallel, for example, 2, 4, 6, 8,10, 12, 16, or 24. Each inlet is situated in a lane of the cartridge,each lane further having channels that divert the respective samples torespective chambers within which an amplification such as PCR can beperformed. The chambers typically can be isolated by one or more valves,during amplification. The chambers are also typically situated so thatthe progress of amplification can be monitored by one or more detectors.Exemplary configurations and manufactures of cartridges are describedelsewhere, including but not limited to U.S. patent application Ser. No.12/173,023, filed on Jul. 14, 2008, and Ser. No. 11/985,577, filed Nov.14, 2007, both of which are incorporated herein by reference.

Typically, the inlet separation on the cartridge, or other receivingarea, is chosen to correspond to the separation between adjacent pipettetips on the dispense heads of the liquid dispenser, or some convenientfraction or multiple thereof. Thus, for example, for a cartridge havingan 8 mm separation between adjacent inlets, used in conjunction with aliquid dispenser having a 24 mm separation between the centers of thetips of adjacent pipette tips, the liquid dispenser can dispense samplesinto cartridge inlets that are separated by two inlets (e.g., a firstand fourth inlets, numbering from a particular end of the cartridge). Itwould be understood that these dimensions and multiples are notlimiting.

The apparatus having been described, it is illustrated by way of thefollowing non-limiting examples.

EXAMPLES Example 1 Exemplary Chemistry and Processes of Use ChemistryOverview

The chemistry processes typically carried out with the apparatusdescribed herein center around the detection and identification oforganisms in a clinical specimen, by virtue of detecting nucleic acidsfrom the organism in question. This involves isolation of nucleic acidsfrom target organisms that are contained in a clinical specimen,followed by a process that will detect the presence of specific nucleicacid sequences. In addition to target detection, an internal positivecontrol nucleic acid can be added to the collection buffer, and canthereby be taken through the entire extraction and detection processalong with target nucleic acids. This control will monitor theeffectiveness of the entire process and will minimize the risk of havingfalse negative results.

Nucleic Acid Extraction and Purification

Nucleic acid extraction procedures begin with the addition of a clinicalspecimen to a prepared specimen collection solution. This can be doneeither at a specimen collection site, or at the testing site. Twocollection solution formats can be available: one for body fluids, andone for swab specimens. Collection solutions used at collection siteswill serve as specimen transport solutions, and therefore, this solutionmust maintain specimen and analyte integrity.

The extraction and purification procedure, which is entirely automatedusing a liquid dispenser as described herein, in conjunction with asuitable heater and separator, proceeds as follows:

-   -   Target organisms are lysed by heating the detergent-containing        collection solution.    -   Magnetic beads, added to the specimen/collection solution mix,        non-specifically bind all DNA that is released into the        solution.    -   Magnetic beads are isolated and are washed to eliminate        contaminants    -   DNA is released from the beads using high pH and heat.    -   DNA containing solution is removed and neutralized with a buffer

Nucleic Acid Amplification

Nucleic acids that have been captured by magnetic beads, washed,released in high pH, and neutralized with buffer, are added to a mixtureof buffers, salts, and enzymes that have been lyophilized in a tube. Themixture is rapidly rehydrated, and then a portion of the solution isloaded onto a microfluidic cartridge. The cartridge is then loaded intothe amplification instrument module, which consists of a heating unitcapable of thermal cycling, and an optical detection system. Detectionof target nucleic acids proceeds as follows:

-   -   The liquid is sealed in a reaction chamber.    -   Rapid thermal cycling is used to potentiate the Polymerase Chain        Reaction (PCR), which is used to amplify specific target DNA.    -   Amplified DNA fluoresces, and can be detected by optical        sensors.    -   A fluorescent probe “tail” is incorporated into each amplified        piece of DNA    -   At a specific temperature, the probe adopts a conformation that        produces fluorescence (this is termed a “scorpion” reaction).    -   Fluorescence is detected and monitored throughout the reaction.

Extraction and Amplification/Detection Process

Extensive bench-scale testing has been performed to optimize the nucleicacid extraction chemistry, including the collection buffer, the washbuffer formulation, the release solution formulation, and the PCRreagent mixes. The fully automated method of extraction, followed by12-up PCR, was able to provide very high sensitivity consistently at 150copies/sample.

Examplary target/sample combinations include: Chlamydia in Urine(50/50); Gonrorrhoea in Urine; GBS in Plasma.

Various detection chemistries such as Taqman, Scorpion, and SYBRg Greenwork reliably in the microfluidic cartridge.

Example 2 Exemplary Chemistry Processes Performed by an AutomatedInstrument Sample Pre-Processing

For Urine Sample: Take 0.5 ml of urine and mix it with 0.5 ml ofcollection buffer. Filter the sample through a pre-filter (containingtwo membranes of 10 micron and 3 micron pore size).

For Plasma Sample: Take 0.5 ml of plasma and mix it with 0.5 ml ofcollection buffer.

For GBS swab samples: Take the swab sample and dip it in 1 ml ofcollection buffer.

For each type of sample, after it is mixed with the appropriatecollection buffer (and filtered if applicable), the solution is placedin the external sample tube in the position specified for it in therack.

The sample collection buffer contains 50 mM Tris pH 7, 1% Triton X-100,20 mM Citrate, 20 mM Borate, 100 mM EDTA, plus 1,000 copies of positivecontrol DNA.

Loading the Instrument and Starting Sample Processing

The following steps may be performed to initiate an analysis on samplesin batch.

-   -   1. Load PCR tube containing PCR master mix in one of the        specified snap-in location of the reagent holder.    -   2. Load PCR tube containing PCR probes and primers for the        target analyte under consideration in the specified location of        the reagent holder.    -   3. In case of two analyte test, load PCR tube containing probes        and primers for second analyte in the specified location of the        reagent holder.    -   4. Insert the reagent holder in a rack, typically a 12-holder        rack, in the same lane as the sample tube under consideration.    -   5. Prepare and insert reagent holders for other samples in        consideration.    -   6. Load the rack in one of the locations in the instrument.    -   7. Load a cartridge in the cartridge tray loading position.        Typically the cartridge has the same number of lanes as the        rack; thus a 12-sample cartridge is used in conjunction with a        12-holder rack.    -   8. Start operation.

Liquid Processing Steps

The following steps may be performed to carry out sample preparation.Herein the numbering of the pipette tips refers to those pipette tipsthat are stored in a reagent holder, for example, in a pipette sheath ofsuch a holder. It would be understood that such operations could beperformed multiply in parallel by a liquid dispenser as describedelsewhere herein. References to a ‘robot’ herein are intended to mean anautomated pipetting apparatus, such as embodiments further describedherein.

-   -   1. Using Pipette tip #1, the robot transfers the clinical sample        from the external sample tube to the process tube of the reagent        holder.    -   2. Using the same pipette tip, the robot takes about 100 μl of        sample, mixes the lyophilized enzyme and affinity beads,        transfers the reagents to the process tube. Mixing is performed        in the process tube by 5 suck and dispense operations.    -   3. The robot places pipette tip #1 at its designated location in        the reagent holder.    -   4. Heat the process tube to 60° C. and maintain it for 10        minutes.    -   5. After 5 minute of lysis, the robot picks up pipette tip #1        and mixes the contents by 3 suck and dispense operations.    -   6. The robot places pipette tip #1 at its designated location in        the reagent holder,    -   7. After 10 minutes of lysis, a magnet is moved up the side of        the process tube to a middle height of the sample and held at        that position for a minute to capture all the magnetic beads        against the wall the tube.    -   8. The magnet is brought down slowly to slide the captured beads        close to the bottom (but not the bottom) of the tube.    -   9. Using pipette tip #2, aspirate all the liquid and dump it        into the waste tube.    -   10. Aspirate a second time to remove as much liquid as possible        from the process tube.    -   11. Using the same pipette tip #2, withdraw 100 μl of wash        buffer and dispense it in the process tube. During this        dispense, the magnet is moved downwards, away from the process        tube.    -   12. Perform 15 mix steps to thoroughly mix the magnetic beads        with the wash buffer.    -   13. Wait for 30 seconds.    -   14. Move magnet up to capture the beads to the side and hold for        15 seconds.    -   15. Using pipette tip #2, aspirate wash buffer twice to remove        as much liquid as possible and dump it back in the wash tube.    -   16. Move magnet down away from the process tube.    -   17. Place pipette tip # 2 in its specified location of the        reagent holder.    -   18. Pick up a new pipette tip (tip #3) and withdraw 8-10 μl of        release buffer and dispense it over the beads in the process        tube.    -   19. Wait for 1 minute and then perform 45 mixes.    -   20. Heat the release solution to 85° C. and maintain temperature        for 5 minutes.    -   21. Place pipette tip # 3 in its specified location of the        reagent holder.    -   22. Bring magnet up the tube, capture all the beads against the        tube wall and move it up and away from the bottom of the tube.    -   23. Pick up a new pipette tip (tip #4) and withdraw all the        release buffer from the process tube and then withdraw 3-10 μl        of neutralization buffer, mix it in the pipette tip and dispense        it in the PCR tube. (In case of two analyte detections, dispense        half of the neutralized DNA solution into first PCR tube and the        rest of the solution in the second PCR tube.)    -   24. Using pipette tip #4, mix the neutralized DNA with the        lyophilized reagents by 4-5 suck and dispense operations and        withdraw the entire solution in the pipette tip.    -   25. Using pipette tip #4, load 6 μl of the final PCR solution in        a lane of the 12-up cartridge.

Real-Time PCR

After all the appropriate PCR lanes of the PCR cartridge are loaded withfinal PCR solution, the tray containing the cartridge moves thecartridge into the PCR Analyzer. The cartridge is pressed by an opticaldetection read-head against the PCR heater. Heaters activate valves toclose either ends of the PCR reactor and the real-time thermocyclingprocess starts. After completing appropriate PCR cycles (˜45 cycles),the analyzer decides whether the sample has the target DNA based on theoutput fluorescence data, and issues an indication of the same.

Example 3 Reagent Holder

An exemplary reagent holder consistent with the description herein hasthe following dimensions and capacities:

-   -   180 mm long×22 mm wide×100 mm tall;    -   Made from Polypropylene.    -   One snapped-in low binding 1.7 ml tube that functions as a        process tube.    -   3 built-in tubes that function as receptacles for reagents, as        follows:        -   One tube containing 200-1000 μl of wash buffer (0.1 mM Tris,            pH 8).        -   One tube containing 200-1000 μl of release solution (40 mM            NaOH).        -   One tube containing 200-1000 μl of neutralization solution            (330 mM Tris, pH 8.0).    -   One built-in tube that functions as a waste chamber (will hold        ˜4 ml of liquid waste).    -   3 receptacles to accept containers for solid reagents. Snap-in        0.3 ml or 0.65 ml PCR tubes (which are typically stored        separately from the reagent holder) are placed in each of these        locations, and contain, respectively:        -   lyophilized sample preparation reagents (lysis enzyme mix            and magnetic affinity beads).        -   First lyophilized PCR master mix, probes and primers for a            first target analyte detection.        -   Second lyophilized PCR master mix, probes and primers for a            second target analyte detection (only offered in select            cases, such as detection of Chlamydia and Gonorrhea from            urine).    -   4 pipette tips located in 4 respective sockets.    -   Pipette tip Sheath: The pipette tips have a sheath/drip tray        underneath to help capture any drip from the pipette tips after        being used, and also to prevent unwanted contamination of the        instrument.    -   Handle and Flex-Lock allows easy insertion, removal, and        positive location of strip in rack.    -   One or more labels: positioned upward facing to facilitate ease        of reading by eye and/or, e.g., a bar-code reader, the one or        more labels containing human and machine readable information        pertaining to the analysis to be performed.

It is to be understood that these dimensions are exemplary. However, itis particularly desirable to ensure that a holder does not exceed thesedimensions so that a rack and an apparatus that accommodates the reagentholder(s) does not become inconveniently large, and can be suitablysituated in a laboratory, e.g., on a bench-top.

Example 4 Exemplary Foil-Sealing of Buffer Containing Reagent Tubes

Tubes containing buffers have to be sealed with high moisture vaporbarrier materials in order to retain the liquid over a long period oftime. Reagent holders may need to have a shelf life of 1-2 years, and assuch, they should not lose more than say 10-15% of the liquid volumeover the time period, to maintain required volume of liquid, and tomaintain the concentration of various molecules present in the solution.Moreover, the materials used for construction of the tube as well as thesealing laminate should not react with the liquid buffer. Specialplastic laminates may provide the moisture barrier but they may have tobe very thick (more than 300 μm thick), causing the piercing force to goup tremendously, or of special, expensive polymer (such as Aclar).Aluminum foils, even a thin foil of a few hundred angstrom provides aneffective moisture barrier but bare aluminum reacts with some liquidbuffers, such as sodium hydroxide, even an aluminum foil with a sprayedcoating of a non-reactive polymer may not be able to withstand thecorrosive vapors over a long time. They may react through tiny pin holespresent in the coating and may fail as a barrier over time.

For these reasons, aluminum foils with a laminate structure have beenidentified as a suitable barrier, exemplary properties of which aredescribed below:

-   -   1. Sealing    -    Heat seals to unitized polypropylene strip (sealing temp        ˜170-180° C.)    -    No wrinkling, cracking and crazing of the foil after sealing    -   2. Moisture Vapor Transmission Rate (MVTR)    -    Loss of less than 10% liquid (20 microliters from a volume of        200 microliter) for a period of 1 year stored at ambient        temperature and pressure. (effective area of transport is ˜63        mm²); Approximate MVTR ˜0.8 cc/m²/day    -   3. Chemistry    -    Ability to not react with 40 mM Sodium Hydroxide (pH<12.6):        foil should have a plastic laminate at least 15 microns thick        closer to the sealed fluid.    -    Ability to not react with other buffers containing mild        detergents    -   4. Puncture    -    Ability to puncture using a p1000 pipette with a force less        than 3 lb    -    Before puncturing, a fully supported membrane 8 mm in diameter        will not stretch more than 5 mm in the orthogonal direction    -    After puncturing, the foil should not seal the pipette tip        around the circumference of the pipette.    -   5. Other Features    -    Pin-hole free    -    No bubbles in case of multi-laminate structures.

Example 5 Illustrative Mechanism of Piercing Through a PlasticizedLaminate and Withdrawing Liquid Buffer

The aluminum laminate containing a plastic film described elsewhereherein serves well for not reacting with corrosive reagents such asbuffers containing NaOH, and having the favorable properties ofpierceability and acting as a moisture barrier. However, it presentssome additional difficulties during piercing. The aluminum foil tends toburst into an irregular polygonal pattern bigger than the diameter ofthe pipette, whereas the plastic film tends to wrap around the pipettetip with minimal gap between the pipette and the plastic film. Thediameter of the hole in the plastic film is similar to the maximumdiameter of the pipette that had crossed through the laminate. Thiswrapping of the pipette causes difficulty in dispensing and pipettingoperations unless there is a vent hole allowing pressures to equilibratebetween outside of the tube and the air inside of the tube.

A strategy for successful pipetting of fluid is as follows:

-   -   1. Pierce through the laminate structure and have the pipette go        close to the bottom of the reagent tube so that the hole created        in the laminate is almost as big as the maximum diameter of the        pipette (e.g., ˜6 mm for a p1000 pipette)    -   2. Withdraw the pipette up a short distance so that a small        annular vent hole is left between the pipette and the laminate.        The p1000 pipette has a smallest outer diameter of 1 mm and        maximum outer diameter of 6 mm and the conical section of the        pipette is about 28 mm long. A vent hole thickness of a hundred        microns is enough to create a reliable vent hole. This        corresponds to the pipette inserted to a diameter of 5.8 mm,        leaving an annulus of 0.1 mm around it.    -   3. Withdraw fluid from the tube. Note that the tube is designed        to hold more fluid than is necessary to withdraw from it for a        typical sample preparation procedure.

Example 6 Exemplary Foil Piercing and Dissolution of LyophilizedReagents

The containers of lyophilized reagents provided in conjunction with aholder as described herein are typically sealed by a non-plasticizedaluminum foil (i.e., not a laminate as is used to seal the reagenttubes). Aluminum foil bursts into an irregular polygonal pattern whenpierced through a pipette and leaves an air vent even though the pipetteis moved to the bottom of the tube. In order to save on reagents, it isdesirable to dissolve the reagents and maximize the amount withdrawnfrom the tube. To accomplish this, a star-ridged (stellated) pattern isplaced at the bottom of the container to maximize liquid volumewithdrawn, and flow velocity in between the ridges.

Exemplary steps for dissolving and withdrawing fluid are as follows:

-   -   1. Pierce through the pipette and dispense the fluid away from        the lyophilized material. If the pipette goes below the level of        the lyophilized material, it will go into the pipette and may        cause jamming of the liquid flow out of the pipette.    -   2. Let the lyophilized material dissolve for a few seconds.    -   3. Move pipette down touching the ridged-bottom of the tube. The        pipette stops moving when it senses an opposition to its motion,        such as by a force sensor described elsewhere herein.    -   4. Perform an adequate number of suck and spit operations (such        as 4-10) to thoroughly mix the reagents with the liquid buffer.    -   5. Withdraw all the reagents and move pipette to dispense it        into the next processing tube.

Example 7 Exemplary Force Sensing of the Pipette Head

Travel of the liquid dispenser along the z-axis is regulated by aforce-sensor. A force sensor is interfaced with the pipette heads insuch a way that any time the pipette head seats against the disposablepipette tip(s) or the picked pipettes are forced through a laminatecover of the reagent holder, or the pipette tip is forced against thebottom of the tubes in the reagent disposable, an upward force acts onthe pipette head through the pipette holding nozzle or the pipette tipitself The entire head is pivoted at a lower point, and any force actingon the head causes a set-screw on the upper part of the head to pressagainst a force sensor. This force sensor is calibrated for verticaldisplacement of the head against a non-moving surface. Using thiscalibration, it can be determined when to stop moving the head in thez-direction by detecting whether, for example, a pipette is properlyseated or if a pipette tip has hit a tube bottom.

Example 8 Exemplary Alignment of Pipette Tips While Loading PCR ReagentSolutions Into a Microfluidic Cartridge

The liquid dispenser is configured so that, when multiple pipette tipsare attached simultaneously, the tips can dispense in parallel tomultiple inlets on a microfluidic cartridge. In particular, this meansthat the spacing between the tips is exactly the same as, or the same asto within an acceptable tolerance, the spacing between the inlets on thecartridge. Larger volume pipette tips can be as long as 95 mm (for,e.g., a p1000 pipette). When 4 long pipette tips are sprung from thehead, even a 1° misalignment during seating can cause the tip to beoff-center by ˜1.7 mm, which is sufficient for that tip to miss thedesired inlet on the cartridge. As it is difficult to have perfectalignment all the time during pipetting of the tip both at its top whereit is interfaced with the tip holder and its bottom, it becomesnecessary to mechanically constrain all the tips at another locationcloser to the bottom. As described elsewhere herein, a stripper platehaving a defined hole structure, can be used to align all the tips. Thestripper plate holes clear all the 4 pipette tips when they are pickedup. After the tips are properly seated, the stripper plate is movedhorizontally, such as in the x-axis direction, using a motor to move allthe pipettes against the notches provided in the stripper plate. Now allthe pipettes land on the cartridge inlet holes with ease.

Example 9 Exemplary Apparatus Including an Automated Pipetting System

Described herein are exemplary specifications for the mechanical designof a system for carrying out PCR on multiple samples. In someembodiments, the system can be about 28.5 inches deep, or less, andabout 43 inches wide, or less, and weight about 250 pounds or less. Thesystem can be designed with a useful life of about 5 years (e.g.,assuming 16,000 tests per year) and can be designed such that the soundlevel for this instrument (during operation) does not exceed 50 dB asmeasured 12 inches from the instrument in all ordinate directions. Insome embodiments, the exterior of the system can be white with texture.

Referring to the overall system, in some embodiments, criticalcomponents of the system can remain orthogonal or parallel (asappropriate) to within 0.04 degrees. Exemplary critical components caninclude motion rails, pipettes, nozzles (e.g., axially as individualnozzles, linearly as an array of four nozzle centroids, or the like),lysis heaters, major edges of the installed cartridge holder in thereader drawer, the front face of the separation magnets, and the like.

In the following descriptions as with elsewhere herein, the X-axis (or Xdirection) refers to the axis extending from left to right when facingthe front of the system, the Y-axis (or Y direction) refers to the axisextending from back to front when facing the front of the system, andthe Z-axis (or Z direction) refers to the axis extending up from thebottom when facing the front of the system. As viewed from the top ofthe instrument, the centroid of the leftmost pipette nozzle on theZ-payload (as viewed from the front of the instrument) can be capable ofunobstructed travel in the X direction from a point 80 mm from theoutermost left baseplate edge to a point 608 mm from the outermost leftbaseplate edge and can be capable of unobstructed travel in the Ydirection from a point 60 mm from the outermost front baseplate edge toa point 410 mm from the outermost front baseplate edge.

Still referring to the system, as viewed from the front of theinstrument, the bottom-most face of the pipette nozzles on the Z-payloadcan be capable of unobstructed travel in the Y direction from a point156 mm above the top surface of the baseplate to a point 256 mm abovethe top surface of the baseplate. The 1 ml pipette tips can be capableof penetrating the foil covers included on disposable reagent strips.This penetration may not create contamination, affect the associatedchemistries, or damage the pipette tips. Motions can be executed in sucha manner as to eliminate mechanical hysteresis, as needed. Gantrymotions can be optimized to prevent cross lane contamination andcarryover. The rack can align the reagent strips to a tolerance of+/−0.010 inches in the X and Y directions.

Referring now to the gantry, in some embodiments, the gantry can consistof a stepper-motor actuated, belt/screw-driven Cartesian robotic system.The gantry can be free to move, with or without attachments, above themodules that are forward of the rear facade and below the bottom-mosthorizontal face on the Z head, so long as the Z-payload is fullyretracted. The gantry can be capable of travel speeds up to about 500mm/sec in the X and Y directions and up to about 100 mm/sec in the Zdirection. The accuracy and precision of the axis motions (e.g., withrespect to the X, Y, and Z home sensors) can be 25 mm or better for eachaxis, and can be retained throughout the maintenance period. The axisdrive belts may not leave residue in areas where PCR and samples areprocessed. The gantry can contain provisions for routing its own and allZ-payload wire harnesses back to the instrument. Belt tension on the Xand Y axes can be set at 41.5+/−3.5 pounds.

Referring now to the Z-payload, the fluid head can have 4 pipetteattachment nozzles located at 24 mm distances between adjacent centers.Such a distance is chosen to facilitate interfacing the pipette tips andinlets on a microfluidic cartridge, as well as between sample tubes, orreagent tubes, on adjacent holders. Exemplary pipette tips that thepipette nozzles can capture without leakage include Biorobotix tipsPN23500048 (50 μL), PN23500049 (1.75 μL), and PN23500046 (1 ml). The Zpayload can incorporate a stepper actuated stripper plate capable ofremoving pipette tips (e.g., the pipette tips described hereinabove).The system can include a pump and manifold system that includes softwarecontrolled aspiration, dispensing, and venting of individual fluidvolumes within each of the four individual tips and simultaneousdispensing and venting on all tips. The pump and manifold system canhave an accuracy and precision of about +/−2 μL per tip for volumes thatare less than 20 μL and about +/−10% for volumes greater than or equalto 20 μL (e.g., when aspirating or dispensing in individual tips). Thetotal pump stroke volume can be greater than about 8 μL and less thanabout 1250 μL. The minimum aspirate and dispense speed can be about 10μL/sec to about 300 μL/sec. The centroid of the bottom-most face of eachpipette tip can be axially aligned with the nozzle centroid of thepipette nozzles within 0.2 mm. The bottom-most pipette tip faces can beco-planar within 0.2 mm. The Z-payload can incorporate a Z axis forcesensor capable of feedback to software for applied forces of betweenabout 0 and 4 lbs. The Z-payload can incorporate a downward facingbarcode reader capable of reading the system barcodes as describedelsewhere herein.

Referring now to racks included in the system, disposable reagent strips(e.g., oriented orthogonally to the front of the instrument) can becontained in 2, 12-lane racks. The 12 reagent strips in a given rack canregister and lock into the rack upon insertion by a user. The rack cancontain an area for 12 sample lysis tubes and hold the tube bottomsco-planar, allowing the user to orient the bar code to face the rear ofthe instrument. Certain features, including those listed above, canallow the racks to be inserted and oriented in the instrument by aminimally trained user. Proper rack placement can be confirmed byfeedback to the software. In some embodiments, the racks can be blackand color fast (e.g, the color may not appreciably degrade with use orwashing with a 10% bleach solution) and the rack material can bedimensionally stable within 0.1 mm over the operating temperature rangeof the system. The rack can be designed with provisions to allow therack to be carried to and from the instrument and to minimize oreliminate the likelihood that the tubes held by the rack will spill whenplaced on a flat surface.

Example 10 Exemplary Pipette Tip Usage

FIGS. 22A-22C show dispense head usage for pipetting operations on banksof 12 samples.

In FIG. 22A, operations on two racks, each containing 12 samples andcorresponding reagent holders (labeled 1-12, and 13-24), are shown. Theleft hand side of the diagram itemizes the set of operations performed.Thus, e.g., “Lysis Prep 1-12” means perform lysis on samples 1-12. Inthis case, it is the same set of operations on each bank of 12 samples.The dashed line (with arrowheads) shows where liquid dispensing head is.Reading the diagram from left to right shows the order of operations.The dispense head can, e.g., alternate between performing operations onthe two racks; the length of a shaded block indicates how long a steptakes. In general, the sequence of operations is set up so that, while,e.g., an processing operation such as heating (that does not require thedispense head) is being carried out on one rack, the dispense head canbe positioned over the other rack and carry out various liquid transferoperations.

FIG. 22B shows details of how one of the steps in FIG. 22A (Lysis prep.)is carried out on 12 samples, as positioned in a single rack. Numbers atthe top of the chart represent time in seconds. The shaded blocks in thegrid indicate the location of the dispense head. The operations areapplied to the samples in batches of 4. Thus, there are 4 distinctoperations to be performed on each sample. In the example shown, acomplete sequence of operations on the first batch of 4 is carried outbefore starting the second batch. It would be understood by one ofordinary skill in the art, that such an approach is exemplary, and thatother sequences of steps, or strategy, could be carried out, consistentwith the overall goal.

FIG. 22C, laid out similarly to FIG. 22B, shows details of sampleremoval, expressed in terms of pipette tip aspiration and dispenseoperations.

The foregoing description is intended to illustrate various aspects ofthe present inventions. It is not intended that the examples presentedherein limit the scope of the present inventions. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A liquid dispenser, comprising: one or more sensors; a manifold; oneor more pumps in fluid communication with the manifold; one or moredispense heads in fluid communication with the manifold; and electricalconnections that accept electrical signals from an external controller,wherein the liquid dispenser has no inlet or outlet for fluids, otherthan through the one or more pumps.
 2. The liquid dispenser of claim 1,having 4 dispense heads, wherein each head is configured to accept apipette tip.
 3. The liquid dispenser of claim 1, configured to aspirateor dispense fluid in connection with analysis or preparation ofsolutions of two or more samples.
 4. The liquid dispenser of claim 1,wherein the one or more sensors comprise a variable force sensor, or astall sensor, that senses interruption in vertical motion of the one ormore dispense heads upon contact with a sample tube or reagent holder.5. The liquid dispenser of claim 1, wherein the one or more dispenseheads are individually sprung.
 6. The liquid dispenser of claim 1,wherein the manifold comprises at least n+1 valves, wherein n is thenumber of dispense heads.
 7. The liquid dispenser of claim 1, wherein nosolution of a sample enters the manifold during normal operation of thedispenser.
 8. The liquid dispenser of claim 1, configured to accept ordispense, in a single operation, an amount of 1.0 ml of fluid or less.9. The liquid dispenser of claim 8, configured to accept or dispense, ina single operation, an amount of fluid in the range 10 nl-1 ml.
 10. Asystem for dispensing and aspirating liquids from one or more sampleholders, the system comprising the liquid dispenser of claim 1, attachedto a gantry that provides the liquid dispenser with freedom oftranslational motion in three dimensions.
 11. The system of claim 10,wherein the translational motion in three dimensions is controlled by amicroprocessor.
 12. The system of claim 10, whereby the controller forthe z-dimension is attached to the pump-manifold system such that thecontroller moves along with the pump.
 13. The system of claim 10,wherein the sensors are selected from the group consisting of:individual pipette tip sensors; an encoder for the pump; a force sensor;and an image sensor.
 14. The system of claim 10, additionally comprisinga barcode sensor.
 15. The system of claim 10, additionally comprising astripper plate to strip pipette tips.
 16. The system of claim 15,additionally comprising a stripper motor with rack and pinion gears tomove the stripper plate in a plane perpendicular to the pipette tips.17. The system of claim 10, where the dispenser dispenses liquid into aprocess tube as well as into a microfluidic cartridge.